Msx homeobox genes provide an ideal model system to address the broad issue of how homeoproteins achieve functional specificity in vivo, since they have been particularly well studied with regard to their expression patterns, biological functions, and biochemical properties. Our extensive analyses of the biochemical properties of Msx homeoproteins have established their functions as transcriptional repressors and have determined that their transcriptional activities are mediated through selective protein-protein interactions. In recent studies, we have been utilizing "discovery" approaches to identify physiologically relevant protein partners for Msx1, as well as downstream target genes. We have established: (i) a yeast two-hybrid screen to isolate Msx1-interacting proteins, (ii) a co-immunoprecipitation approach to isolate Msx1-protein complexes from living cells, and (iii) a microarray approach to identify differentially regulated genes. These strategies have led to three key observations: (i) that the activity of Msx1 is regulated by post-translational modification by sumoylation; (ii) that histone H1, a transcriptional repressor, interacts with Msx1 in living cells; and (iii) that genes which are differentially regulated by Msx1 include those involved in cellular differentiation. These "discovery" approaches serve as the basis for our proposed hypothesis-driven experiments, in which we will address the complex issue of how Msx homeoproteins achieve functional specificity in vivo by integrating a variety of biochemical and biological approaches. Our hypothesis is that the functional specificity of Msx homeoproteins in vivo represents the outcome of a complex interplay of several components, including post-translational modifications, protein-protein interactions, and cellular context. Thus, in Specific Aim 1, we will investigate the functional consequences of post-translational modification by sumoylation through our analysis of the biochemical properties, sub-cellular distribution, and biological activities of the sumoylated and non-sumoylated forms of Msx1. In Specific Aim 2, we will analyze protein partners for Msx1 in living cells by investigating the biochemical and biological consequences of Msx1-histone H1 interaction, focusing on their mutual ability to repress target genes, such as MyoD. These studies will provide a framework for our subsequent analysis of endogenous Msx1-protein complexes in living cells and embryos, which will be pursued using an epitope-tagged Msx1 "knock-in" allele. In Specific Aim 3, we will use a tamoxifen-inducible expression system (Msx1-ERtm) to identify direct target genes for Msx1, which will facilitate the subsequent identification of physiologically relevant "Msx1-responsive elements." Finally, in Specific Aim 4, we will analyze the limb phenotype of conventional and conditional mutant mice to address the biological functions of Msx genes in the limb. Taken together, our focused analyses of Msx homeoproteins will provide general paradigms for addressing the functional specificities of other homeoproteins in vivo.